klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Tue Mar 30 20:14:57 EST 1999
: Has anybody used Streptolysin O for permeabilizing some cells? I'm trying
:to permeabilize mast cells with SLO. And an unexpected
:problem came up. I don't know
:how to store the reagent in stable state. I bought it from Sigma and
:dissolved with water, aliquoted, and froze it. And just before using
:it, I treated it with 20mM of DTT to activate it because SLO is readily
:oxidized in solution, which is inactive . On the first day I got
:my cells permeabilized by SLO treatment. But from the next day, I have
:got the same result even though I used ten times higher concentration of
:SLO. Sigma says SLO solution should be made fresh , but it's almost
:impossible because one bottle has only 900ug. This is frustrating. Does
:anybody have an answer to this problem? Please let me know ASAP, please.
: Thanks in advance.
Use digitonin permeabilization :-)
SLO is just too irreproducible even, IMHO, in the labs where it is used
routinly. Or, if your assay would survive it, you can use "cracked" cells
approach where permeable cell ghosts free of cytosol are prepared
first - this one is incredibly reproducible and robust.
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